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The cerebral cortex forms early in development according to a series of heritable neurodevelopmental instructions. Despite deep evolutionary conservation of the cerebral cortex and its foundational six-layered architecture, significant variations in cortical size and folding can be found across mammals, including a disproportionate expansion of the prefrontal cortex in humans. Yet our mechanistic understanding of neurodevelopmental processes is derived overwhelmingly from rodent models, which fail to capture many human-enriched features of cortical development. With the advent of pluripotent stem cells and technologies for differentiating three-dimensional cultures of neural tissue in vitro, cerebral organoids have emerged as an experimental platform that recapitulates several hallmarks of human brain development. In this review, we discuss the merits and limitations of cerebral organoids as experimental models of the developing human brain. We highlight innovations in technology development that seek to increase its fidelity to brain development in vivo and discuss recent efforts to use cerebral organoids to study regeneration and brain evolution as well as to develop neurological and neuropsychiatric disease models. [ABSTRACT FROM AUTHOR]

Background: High throughput methods for profiling the transcriptomes of single cells have recently emerged as transformative approaches for large-scale population surveys of cellular diversity in heterogeneous primary tissues. However, the efficient generation of such atlases will depend on sufficient sampling of diverse cell types while remaining cost-effective to enable a comprehensive examination of organs, developmental stages, and individuals. Results: To examine the relationship between sampled cell numbers and transcriptional heterogeneity in the context of unbiased cell type classification, we explored the population structure of a publicly available 1.3 million cell dataset from E18.5 mouse brain and validated our findings in published data from adult mice. We propose a computational framework for inferring the saturation point of cluster discovery in a single-cell mRNA-seq experiment, centered around cluster preservation in downsampled datasets. In addition, we introduce a "complexity index," which characterizes the heterogeneity of cells in a given dataset. Using Cajal-Retzius cells as an example of a limited complexity dataset, we explored whether the detected biological distinctions relate to technical clustering. Surprisingly, we found that clustering distinctions carrying biologically interpretable meaning are achieved with far fewer cells than the originally sampled, though technical saturation of rare populations such as Cajal-Retzius cells is not achieved. We additionally validated these findings with a recently published atlas of cell types across mouse organs and again find using subsampling that a much smaller number of cells recapitulates the cluster distinctions of the complete dataset. Conclusions: Together, these findings suggest that most of the biologically interpretable cell types from the 1.3 million cell database can be recapitulated by analyzing 50,000 randomly selected cells, indicating that instead of profiling few individuals at high "cellular coverage," cell atlas studies may instead benefit from profiling more individuals, or many time points at lower cellular coverage and then further enriching for populations of interest. This strategy is ideal for scenarios where cost and time are limited, though extremely rare populations of interest (< 1%) may be identifiable only with much higher cell numbers. [ABSTRACT FROM AUTHOR]

Haldipur et al. explored the developmental origins of the human cerebellum, which has gained growing appreciation for its involvement in human cognition. The authors discovered human-unique expansion and maintenance of cerebellar germinal zones, reminiscent of processes in the developing human cerebral cortex necessary for generating expanded neuronal populations. [ABSTRACT FROM AUTHOR]

Systematic analyses of spatiotemporal gene expression trajectories during organogenesis have been challenging because diverse cell types at different stages of maturation and differentiation coexist in the emerging tissues. We identified discrete cell types as well as temporally and spatially restricted trajectories of radial glia maturation and neurogenesis in developing human telencephalon. These lineage-specific trajectories reveal the expression of neurogenic transcription factors in early radial glia and enriched activation of mammalian target of rapamycin signaling in outer radial glia. Across cortical areas, modest transcriptional differences among radial glia cascade into robust typological distinctions among maturing neurons. Together, our results support a mixed model of topographical, typological, and temporal hierarchies governing cell-type diversity in the developing human telencephalon, including distinct excitatory lineages emerging in rostral and caudal cerebral cortex. [ABSTRACT FROM AUTHOR]

Summary The classic view of cortical development, embodied in the radial unit hypothesis, highlights the ventricular radial glia (vRG) scaffold as a key architectonic feature of the developing neocortex. The scaffold includes continuous fibers spanning the thickness of the developing cortex during neurogenesis across mammals. However, we find that in humans, the scaffold transforms into a physically discontinuous structure during the transition from infragranular to supragranular neuron production. As a consequence of this transformation, supragranular layer neurons arrive at their terminal positions in the cortical plate along outer radial glia (oRG) cell fibers. In parallel, the radial glia that contact the ventricle develop distinct gene expression profile and “truncated” morphology. We propose a supragranular layer expansion hypothesis that posits a deterministic role of oRG cells in the radial and tangential expansion of supragranular layers in primates, with implications for patterns of neuronal migration, area patterning, and cortical folding. [ABSTRACT FROM AUTHOR]

Summary Long non-coding RNAs (lncRNAs) are a diverse and poorly conserved category of transcripts that have expanded greatly in primates, particularly in the brain. We identified an lncRNA, which has acquired 16 microRNA response elements for miR-143-3p in the Catarrhini branch of primates. This lncRNA, termed LncND (neurodevelopment), is expressed in neural progenitor cells and then declines in neurons. Binding and release of miR-143-3p by LncND control the expression of Notch receptors. LncND expression is enriched in radial glia cells (RGCs) in the ventricular and subventricular zones of developing human brain. Downregulation in neuroblastoma cells reduced cell proliferation and induced neuronal differentiation, an effect phenocopied by miR-143-3p overexpression. Gain of function of LncND in developing mouse cortex led to an expansion of PAX6+ RGCs. These findings support a role for LncND in miRNA-mediated regulation of Notch signaling within the neural progenitor pool in primates that may have contributed to the expansion of cerebral cortex. [ABSTRACT FROM AUTHOR]

Summary Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC , PTPRZ1 , FAM107A , HOPX , and LIFR. Using dynamic imaging, immunostaining, and clonal analysis, we relate these molecular features to distinctive behaviors of outer radial glia, demonstrate the necessity of STAT3 signaling for their cell cycle progression, and establish their extensive proliferative potential. These results suggest that outer radial glia directly support the subventricular niche through local production of growth factors, potentiation of growth factor signals by extracellular matrix proteins, and activation of self-renewal pathways, thereby enabling the developmental and evolutionary expansion of the human neocortex. [ABSTRACT FROM AUTHOR]

Evolutionary expansion of the human neocortex underlies many of our unique mental abilities. This expansion has been attributed to the increased proliferative potential of radial glia (RG; neural stem cells) and their subventricular dispersion from the periventricular niche during neocortical development. Such adaptations may have evolved through gene expression changes in RG. However, whether or how RG gene expression varies between humans and other species is unknown. Here we show that the transcriptional profiles of human and mouse neocortical RG are broadly conserved during neurogenesis, yet diverge for specific signalling pathways. By analysing differential gene co-expression relationships between the species, we demonstrate that the growth factor PDGFD is specifically expressed by RG in human, but not mouse, corticogenesis. We also show that the expression domain of PDGFRβ, the cognate receptor for PDGFD, is evolutionarily divergent, with high expression in the germinal region of dorsal human neocortex but not in the mouse. Pharmacological inhibition of PDGFD-PDGFRβ signalling in slice culture prevents normal cell cycle progression of neocortical RG in human, but not mouse. Conversely, injection of recombinant PDGFD or ectopic expression of constitutively active PDGFRβ in developing mouse neocortex increases the proportion of RG and their subventricular dispersion. These findings highlight the requirement of PDGFD-PDGFRβ signalling for human neocortical development and suggest that local production of growth factors by RG supports the expanded germinal region and progenitor heterogeneity of species with large brains. [ABSTRACT FROM AUTHOR]

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (∼50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells. [ABSTRACT FROM AUTHOR]

• The human brain is unique from other species. • Brain organogenesis is a complex and highly orchestrated developmental process. • Radial glial cells give rise to the neuronal diversity of the adult brain. • Neuronal populations organize into complex cytoarchitectural patterns. • Cerebral organoids are derived from human pluripotent stem cells. • Organoids self-organize and develop the major cell classes found in the brain. • The neurogenic to gliogenic transition is well represented in organoids. • The fidelity of circuit formation and maturation in organoids is unclear. • More data-driven approaches for unbiased comparisons of organoid are required. The brain is one of the most complex organs in the body, which emerges from a relatively simple set of basic 'building blocks' during early development according to complex cellular and molecular events orchestrated through a set of inherited instructions. Innovations in stem cell technologies have enabled modelling of neural cells using two- and three-dimensional cultures. In particular, cerebral ('brain') organoids have taken the center stage of brain development models that have the potential for providing meaningful insight into human neurodevelopmental and neurological disorders. We review the current understanding of cellular events during human brain organogenesis, and the events occurring during cerebral organoid differentiation. We highlight the strengths and weaknesses of this experimental model system. In particular, we explain evidence that organoids can mimic many aspects of early neural development, including neural induction, patterning, and broad neurogenesis and gliogenesis programs, offering the opportunity to study genetic regulation of these processes in a human context. Several shortcomings of the current culture methods limit the utility of cerebral organoids to spontaneously give rise to many important cell types, and to model higher order features of tissue organization. We suggest that future studies aim to improve these features in order to make them better models for the study of laminar organization, circuit formation and how disruptions of these processes relate to disease. [ABSTRACT FROM AUTHOR]

Adeno-associated viruses (AAVs) hold tremendous promise as delivery vectors for gene therapies. AAVs have been successfully engineered--for instance, for more efficient and/or cell-specific delivery to numerous tissues--by creating large, diverse starting libraries and selecting for desired properties. However, these starting libraries often contain a high proportion of variants unable to assemble or package their genomes, a prerequisite for any gene delivery goal. Here, we present and showcase a machine learning (ML) method for designing AAV peptide insertion libraries that achieve fivefold higher packaging fitness than the standard NNK library with negligible reduction in diversity. To demonstrate our ML-designed library's utility for downstream engineering goals, we show that it yields approximately 10-fold more successful variants than the NNK library after selection for infection of human brain tissue, leading to a promising glial-specific variant. Moreover, our design approach can be applied to other types of libraries for AAV and beyond. [ABSTRACT FROM AUTHOR]

Most genetic studies consider autism spectrum disorder (ASD) and developmental disorder (DD) separately despite overwhelming comorbidity and shared genetic etiology. Here, we analyzed de novo variants (DNVs) from 15,560 ASD (6,557 from SPARK) and 31,052 DD trios independently and also combined as broader neurodevelopmental disorders (NDDs) using three models. We identify 615 NDD candidate genes (false discovery rate [FDR] < 0.05) supported by ≥1 models, including 138 reaching Bonferroni exome-wide significance (P < 3.64e-7) in all models. The genes group into five functional networks associating with different brain developmental lineages based on single-cell nuclei transcriptomic data. We find no evidence for ASD-specific genes in contrast to 18 genes significantly enriched for DD. There are 53 genes that show mutational bias, including enrichments for missense (n = 41) or truncating (n = 12) DNVs. We also find 10 genes with evidence of male- or female-bias enrichment, including 4 X chromosome genes with significant female burden (DDX3X, MECP2, WDR45, and HDAC8). This large-scale integrative analysis identifies candidates and functional subsets of NDD genes. [ABSTRACT FROM AUTHOR]

Progenitors of the developing human neocortex reside in the ventricular and outer subventricular zones (VZ and OSVZ, respectively). However, whether cells derived from these niches have similar developmental fates is unknown. By performing fate mapping in primary human tissue, we demonstrate that astrocytes derived from these niches populate anatomically distinct layers. Cortical plate astrocytes emerge from VZ progenitors and proliferate locally, while putative white matter astrocytes are morphologically heterogeneous and emerge from both VZ and OSVZ progenitors. Furthermore, via single-cell sequencing of morphologically defined astrocyte subtypes using Patch-seq, we identify molecular distinctions between VZ-derived cortical plate astrocytes and OSVZ-derived white matter astrocytes that persist into adulthood. Together, our study highlights a complex role for cell lineage in the diversification of human neocortical astrocytes. [ABSTRACT FROM AUTHOR]

The article offers information on using lentivirus-based massively parallel reporter assays (lentiMPRAs) combined with deep learning to evaluate candidate gene regulatory elements. Topics include the identification of sequences exhibiting enhancer activity; the discovery of variants associated with psychiatric disorders showing differential enhancer activity; and the training of a deep learning model to predict nucleotide changes' effects on enhancer function.

Background: Embryonic stem (ES) cells can differentiate into all cell types and have been used extensively to study factors affecting neuronal differentiation. ES cells containing mutations in known genes have the potential to provide useful in vitro models for the study of gene function during neuronal differentiation. Recently, mouse ES cell lines lacking the neurogenic transcription factor Pax6 were reported; neurons derived from these Pax6-/- ES cells died rapidly after neuronal differentiation in vitro. Results: Here we report the derivation of new lines of Pax6-/- ES cells and the assessment of their ability to survive and differentiate both in vitro and in vivo. Neurons derived from our new Pax6-/- lines were viable and continued to elaborate processes in culture under conditions that resulted in the death of neurons derived from previously reported Pax6-/- ES cell lines. The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo. We used our new lines to generate Pax6-/- ↔ Pax6+/+ chimeras in which the mutant cells survived and displayed the same phenotypes as Pax6-/- cells in Pax6-/- ↔ Pax6+/+ chimeras made by embryo aggregation. Conclusions: We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons. We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development. [ABSTRACT FROM AUTHOR]

Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods. Ly et al. report a multi-well imaging system (the Picroscope) that can perform longitudinal bright-field z-stack imaging for 24 wells simultaneously. They demonstrate its capability by imaging zebrafish embryos, planaria worms, development of Xenopus tropicalis over a 28-h period and embryonic stem cell monolayer and organoids inside an incubator. [ABSTRACT FROM AUTHOR]

Summary As the use of single-cell technologies has grown, so has the need for tools to explore these large, complicated datasets. The UCSC Cell Browser is a tool that allows scientists to visualize gene expression and metadata annotation distribution throughout a single-cell dataset or multiple datasets. Availability and implementation We provide the UCSC Cell Browser as a free website where scientists can explore a growing collection of single-cell datasets and a freely available python package for scientists to create stable, self-contained visualizations for their own single-cell datasets. Learn more at https://cells.ucsc.edu. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Neuroserpin is a serine‐protease inhibitor mainly expressed in the CNS and involved in the inhibition of the proteolytic cascade. Animal models confirmed its neuroprotective role in perinatal hypoxia‐ischaemia and adult stroke. Although neuroserpin may be a potential therapeutic target in the treatment of the aforementioned conditions, there is still no information in the literature on its distribution during human brain development. The present study provides a detailed description of the changing spatiotemporal patterns of neuroserpin focusing on physiological human brain development. Five stages were distinguished within our examined age range which spanned from the 7th gestational week until adulthood. In particular, subplate and deep cortical plate neurons were identified as the main sources of neuroserpin production between the 25th gestational week and the first postnatal month. Our immunohistochemical findings were substantiated by single cell RNA sequencing data showing specific neuronal and glial cell types expressing neuroserpin. The characterization of neuroserpin expression during physiological human brain development is essential for forthcoming studies which will explore its involvement in pathological conditions, such as perinatal hypoxia‐ischaemia and adult stroke in human. [ABSTRACT FROM AUTHOR]

The article discusses a study on the spatiotemporal molecular dynamics of the developing human thalamus, conducted by Chang N. Kim and David Shin, and published in the journal "Nature Neuroscience." The study provides a comprehensive characterization of cell types and their spatial organization in the developing human thalamus, revealing the differentiation of glutamatergic and GABAergic neurons, expansion of GABAergic neurons from outside brain regions, and the onset of gliogenesis.

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme ( GBM) is the most common and aggressive type of primary brain tumor. Epidermal growth factor ( EGF) and platelet-derived growth factor ( PDGF) receptors are frequently amplified and/or possess gain-of-function mutations in GBM. However, clinical trials of tyrosine-kinase inhibitors have shown disappointing efficacy, in part due to intra-tumor heterogeneity. To assess the effect of clonal heterogeneity on gene expression, we derived an approach to map single-cell expression profiles to sequentially acquired mutations identified from exome sequencing. Using 288 single cells, we constructed high-resolution phylogenies of EGF-driven and PDGF-driven GBMs, modeling transcriptional kinetics during tumor evolution. Descending the phylogenetic tree of a PDGF-driven tumor corresponded to a progressive induction of an oligodendrocyte progenitor-like cell type, expressing pro-angiogenic factors. In contrast, phylogenetic analysis of an EGFR-amplified tumor showed an up-regulation of pro-invasive genes. An in-frame deletion in a specific dimerization domain of PDGF receptor correlates with an up-regulation of growth pathways in a proneural GBM and enhances proliferation when ectopically expressed in glioma cell lines. In-frame deletions in this domain are frequent in public GBM data. [ABSTRACT FROM AUTHOR]

Background: The mammalian thalamus relays sensory information from the periphery to the cerebral cortex for cognitive processing via the thalamocortical tract. The thalamocortical tract forms during embryonic development controlled by mechanisms that are not fully understood. β-catenin is a nuclear and cytosolic protein that transduces signals from secreted signaling molecules to regulate both cell motility via the cytoskeleton and gene expression in the nucleus. In this study we tested whether β-catenin is likely to play a role in thalamocortical connectivity by examining its expression and activity in developing thalamic neurons and their axons.Results: At embryonic day (E)15.5, the time when thalamocortical axonal projections are forming, we found that the thalamus is a site of particularly high β-catenin mRNA and protein expression. As well as being expressed at high levels in thalamic cell bodies, β-catenin protein is enriched in the axons and growth cones of thalamic axons and its growth cone concentration is sensitive to Netrin-1. Using mice carrying the β-catenin reporter BAT-gal we find high levels of reporter activity in the thalamus. Further, Netrin-1 induces BAT-gal reporter expression and upregulates levels of endogenous transcripts encoding β-actin and L1 proteins in cultured thalamic cells. We found that β-catenin mRNA is enriched in thalamic axons and its 3'UTR is phylogenetically conserved and is able to direct heterologous mRNAs along the thalamic axon, where they can be translated.Conclusion: We provide evidence that β-catenin protein is likely to be an important player in thalamocortcial development. It is abundant both in the nucleus and in the growth cones of post-mitotic thalamic cells during the development of thalamocortical connectivity and β-catenin mRNA is targeted to thalamic axons and growth cones where it could potentially be translated. β-catenin is involved in transducing the Netrin-1 signal to thalamic cells suggesting a mechanism by which Netrin-1 guides thalamocortical development. [ABSTRACT FROM AUTHOR]

Parallel in vivo analysis of large-effect autism genes implicates cortical neurogenesis and estrogen in risk and resilience. [Extracted from the article]

Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks. • In vivo analysis of autism genes reveals a convergent vulnerability in neurogenesis • Systems biological analysis implicates the inner cortical plate and subventricular zone • Estrogen can mitigate the effects of disparate autism gene mutations • Estrogen inhibits Sonic hedgehog signaling Using parallel in vivo analyses and systems biological approaches, Willsey et al. implicate cortical neurogenesis as a point of convergent vulnerability in autism spectrum disorders. They identify estrogen as a resilience factor for multiple, disparate autism genes and reveal a conserved role for estrogen in repressing Sonic hedgehog signaling. [ABSTRACT FROM AUTHOR]

To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1 , and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease. • ∼19,000 enhancers defined in nine regions of the developing human telencephalon • Chromatin dynamics correlate with sequence motifs and spatiotemporal gene expression • Identified cortical layer-specific enhancers and validated a layer 5 FEZF2 enhancer • Genetic variants from patients alter activity of an enhancer for an autism risk gene A high-resolution atlas of regulatory elements driving regional, temporal, and laminar gene expression programs in the developing human telencephalon reveals enhancers and genetic variants regulating human disease genes. [ABSTRACT FROM AUTHOR]

Current approaches for dynamic profiling of single cells rely on dissociated cultures, which lack important biological features existing in tissues. Organotypic slice cultures preserve aspects of structural and synaptic organisation within the brain and are amenable to microscopy, but established techniques are not well adapted for high throughput or longitudinal single cell analysis. Here we developed a custom-built, automated confocal imaging platform, with improved organotypic slice culture and maintenance. The approach enables fully automated image acquisition and four-dimensional tracking of morphological changes within individual cells in organotypic cultures from rodent and human primary tissues for at least 3 weeks. To validate this system, we analysed neurons expressing a disease-associated version of huntingtin (HTT586Q138-EGFP), and observed that they displayed hallmarks of Huntington's disease and died sooner than controls. By facilitating longitudinal single-cell analyses of neuronal physiology, our system bridges scales necessary to attain statistical power to detect developmental and disease phenotypes. Jeremy Linsley, Atmiyata Tripathi et al. optimise culturing techniques and develop a microscopy platform and that can automatically image and track single neurons within organotypic slices for up to a three-week period. Using a model of polyQ neurodegeneration, they show that this method can assess longitudinal cell changes with high accuracy. [ABSTRACT FROM AUTHOR]

Background: Embryonic stem (ES) cells can differentiate into all cell types and have been used extensively to study factors affecting neuronal differentiation. ES cells containing mutations in known genes have the potential to provide useful in vitro models for the study of gene function during neuronal differentiation. Recently, mouse ES cell lines lacking the neurogenic transcription factor Pax6 were reported; neurons derived from these Pax6-/- ES cells died rapidly after neuronal differentiation in vitro.Results: Here we report the derivation of new lines of Pax6-/- ES cells and the assessment of their ability to survive and differentiate both in vitro and in vivo. Neurons derived from our new Pax6-/- lines were viable and continued to elaborate processes in culture under conditions that resulted in the death of neurons derived from previously reported Pax6-/- ES cell lines. The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo. We used our new lines to generate Pax6-/- <--> Pax6+/+ chimeras in which the mutant cells survived and displayed the same phenotypes as Pax6-/- cells in Pax6-/- <--> Pax6+/+ chimeras made by embryo aggregation.Conclusions: We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons. We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development. [ABSTRACT FROM AUTHOR]